Reprod Dom Anim 46, 165–172 (2011); doi: 10.1111/j.1439-0531.2010.01603.
x
ISSN 0936-6768
Review Article
Objective Sperm Motion Analysis to Assess Dairy Bull Fertility Using
Computer-Aided System – A Review
P Kathiravan1, J Kalatharan2, G Karthikeya2, K Rengarajan3 and G Kadirvel4
1
National Bureau of Animal Genetic Resources, Karnal, Haryana; 2Department of AGB, Madras Veterinary College, Chennai, Tamilnadu; 3National
Dairy Research Institute, Karnal, Haryana; 4ICAR Research Complex for NEH Region, Umiam, Megalaya, India
Contents
Motility is one of the most important characteristics associated
with the fertilizing ability of spermatozoa and is an expression
of their viability and structural integrity. Computer-assisted
semen analyser (CASA) provides precise and accurate infor-
mation on different sperm motion characteristics. This article
reviews various aspects of computer-aided motility analysis of
bull sperm like sample preparation, standardization of instru-
ment settings, importance of various motility parameters
evaluated by the system and its impact on basic functional
studies of spermatozoa. It gives special emphasis to various
aspects of bull sperm motion analysis especially sub-popula-
tions of spermatozoa, hyper-activation, motion characteristic
in different genetic and age groups, etc. and their utility in
predicting the fertility of dairy bulls. The need to fill the gap in
research and the necessity of universal standardization of the
equipment has been discussed.
Introduction
Male fertility is an important factor in bovine repro-
duction as a single bull is generally used to breed
numerous cows especially after the introduction of
artificial insemination (AI) technology. Assessment of
male fertility is primarily based on post-thaw semen
evaluation using conventional parameters such as sperm
motility, morphology, viability, biochemical estimations
of enzyme release, membrane and acrosome integrity.
Among these, motility is commonly believed to be one
of the most important characteristics associated with the
fertilizing ability of spermatozoa. Motility remains the
parameter of choice to determine the degree of sperm
damage inflicted by the cryopreservation procedure.
Sperm motility is widely evaluated by visual assessment
on a microscope equipped with phase contrast optics.
However, because of the subjectivity of the technique, it
has been a poor predictor of fertility potential (Liu et al.
1988; Fitzpatrick et al. 2002; Holroyd et al. 2002).
Numerous attempts were made for objective evalua-
tion of sperm motility. ‘Track motility’ was first
suggested by Rothschild (1953) for evaluating sperm
motility. Laser-doppler spectroscopy (Budworth et al.
1987); turbidimetry (Donnelly et al. 1998) and photo-
metric methods (Burkman 1991) were developed, but
these systems allowed crude estimation of the whole
population, without taking into account of the evalua-
tion of individual spermatozoa. Computer-aided semen
analysis was first proposed by Dott and Foster (1971) in
which several parameters like concentration, motility
 2010 Blackwell Verlag GmbH
and morphology can be analysed by processing digital
images of the spermatozoa. Computer-assisted semen
analysers (CASA) provide precise and accurate infor-
mation on different sperm motion characteristics
(Gravance and Davis 1995; Holt and Palomo 1996).
These computer-aided systems analyse the spermatozoa
for various motility characteristics such as progressive
motility, track speed, progressive velocity, path velocity,
linearity, amplitude of lateral head displacement, beat
cross frequency (BCF). Artificial insemination stations
are now adopting CASAs to increase objectivity in
determination of sperm motility.
Brief Description of CASA and its Function
The CASA system commonly consists of a microscope
attached to a video camera, a video frame grabber card
and a computer. Spermatozoa are normally perceived
using a dark field ⁄ negative phase contrast ⁄ fluorescent
optics microscope. In dark field, negative phase contrast
microscope, white sperm heads are visualized on a dark
background and the brightness of sperm head is utilized
to establish centroid positions in successive fields. In
fluorescent optics microscope, sperm head is identified
by staining with a fluorescent dye that binds to the
sperm DNA. Recently, light microscope has been tried
for CASA system with image enhancement and a special
sperm tracking algorithm (Nafisi et al. 2005). The video
camera captures the microscopic images of the sperma-
tozoa which is then digitized by the computer based on
number of picture elements (pixels) covered by the
sperm head. The user can define the pixel range covered
by sperm heads for different species. To reduce the error
as a result of debris in the given pixel range, different
CASA systems use different approaches. Some require
an attached tail with an identified sperm head (Strom-
berg-Mika CASA system; Neuwinger et al. 1990;
Wijchman et al. 1995), while others use the IDENT
stain in which the fluorescent dye binding to DNA in
sperm head is recognized by the computer. However, the
main limitation of using the fluorescent dye to differen-
tiate the sperm head from debris is that the sperm must
be static in position during analysis. Hence, this cannot
be used for estimating the motility parameters although
it is proved to be much useful for estimating the sperm
concentration (Zinaman et al. 1996).
The number of microscopic images obtained per
second in a CASA system depends on the picture
166 P Kathiravan, J Kalatharan, G Karthikeya, K Rengarajan and G Kadirvel

standard of the video camera used. Most systems use
standard video image acquisition rates typically 50 Hz
[phase alternating line (PAL) standard] or 60 Hz
[National Television Standards Committee (NTSC)
standard]. After digitization of sperm heads in successive
frames, a path-finding algorithm (Katz and Davis 1987)
is used to identify and track the progress of spermatozoa
across the field of view. The algorithm used differs
among various CASA systems, but the basic principle of
the steps involved are as follows. The first step involves
establishing the centroid positions of the sperm head in
successive fields by estimating the likelihood or zone of
probability that a spermatozoon could travel (Mortimer
2000). The second step in the algorithm establishes the
number of centroids to be analysed for a sperm
trajectory based on the minimum time interval allotted.
The third step specifies the minimum average distance
between successive video fields that a spermatozoon
must translate to continue to be considered as moving.
In the final step, the algorithm specifies the number of
forward video fields to be looked for regaining contact
with the path of missing centroid. The trajectory of the
sperm movement is thus reconstructed and a series of
kinematic parameters are estimated.
The process of reconstructing the sperm track from
successive images is crucial for the accuracy of the
CASA system and it depends on (i) spatial resolution of
magnification systems (microscope and camera charac-
teristics), (ii) the temporal resolution of the systems i.e.
frame rate in relation to speed of the sperm head
(Acosta and Kruger 1996), (iii) size of the sperm head
and (iv) mistaken path of a sperm because of collisions.
In the later case, standardization of sperm concentration
of the samples plays a major role as there is a risk of
having errors in the results or removing such collided
tracks from analysis (Mortimer 2000).
Sperm Motion Analysis by CASA
Instrument settings for bull sperm analysis
Computer-assisted semen analysis facilitates objective
classification of sperm population. CASA machines
process the algorithms and analyse motion properties of
spermatozoa based on their digital images. The users
can select the frame acquisition rate (number of frames
per second analysed) and the total number of analysed
frames so that sperm cells can be successfully tracked for
their inclusion in the motion analysis. Users can also
define different set-ups (intensity, size values), which are
essential to discriminate between sperm heads from
debris ⁄ other cells present in the sample being evaluated.
Instrument settings like gates in analysing the specimen,
number of fields and samples examined, temperature at
which measures are realized, the method of processing
semen for CASA and many other factors can dramat-
ically influence the results (Davis and Katz 1993;
Mortimer 1997). The set-up needs to be evaluated for
different species, in the full range of clinical situations as
there is considerable heterogeneity of sperm size and
motility. The equipment has to be standardized for a
considered species before determining the precision and
accuracy of the system. Owen and Katz (1993) suggested
60 frames per second for CASA applications in human
and mammalian semen analysis. Different authors have
tried different parameters set up of CASA for analysing
bull spermatozoa with different extenders (Table 1). The
reliability, accuracy and precision of CASA analysis
under various experimental conditions depend largely
on the expertise and training of the users on certain
aspects like calibrations, validation, standardization and
optimization of the semen processing before analysis
(Verstegen et al. 2002).
Sample preparation for CASA analysis
Preparation of semen samples for analysis in CASA is
critical from three aspects; sperm concentration, ex-
tender used to dilute semen and loading volume used for
analysis. Dilution of semen sample is essential to adjust
the concentration for successful analysis of individual
sperm tracks. The concentration of sperms used for
analysis is significant to obtain accurate kinetic mea-
surements as higher concentrations can cause multiple
individual collisions of sperm cells and falsify the
motility results (Rijsselaere et al. 2002). The extender
used to dilute the sample must not contain particles of

Table 1. Different settings of computer-assisted semen analyser for bull sperm analysis

Parameter ⁄ medium used TEYG SCC TALP CUE TALP TEYG Phy Saline

Frames acquired – – 30 30 30 30 30
Frame rate (Hz) 7 6 60 60 30 60 60
Minimum contrast 15 15 30 20 8 60 20
Minimum cell size (pixels) – – 4 5 8 5 10
Threshold straightness (%) – – 80 80 60 64 70
Low VAP cut-off (lm ⁄ s) – – 25 75 20 15 30
Medium VAP cut-off (lm ⁄ s) – – 75 35 80 80 –
Head size, Non-motile (pixels) – – 5 7 13 7 5
Head Intensity, Non-motile – – 55 30 21 80 20
Static head size (pixels) 0.6–3.0 0.6–3.0 0.7–2.75 0.85–2.40 0.40–1.6 0.70–1.95 –
Static head intensity 0.5–3.8 0.5–3.8 0.3–2.0 0.40–1.80 0.30–1.6 0.41–1.44 0.50–3.0
Static elongation (pixels) – – 0–65 0–60 – 16–84 0–85
Temperature (C) – – 37 37 37 37 37
Reference Anzar et al. (1991) Tardif et al. (1997) Farrell et al. Kathiravan Hoflack
(1998) et al. (2005) et al. (2007)

TEYG, tris-egg yolk- glycerol; SCC, sodium citrate-casein; TALP, Tyrode’s albumin-lactate-pyruvate; CUE, Cornell University extender; Phy saline, physiological
saline, VAP, average path velocity.

 2010 Blackwell Verlag GmbH

Motion Analysis of Bull Sperm in CASA – A Review
size similar to sperm heads (Anzar et al. 1991). Various
extenders such as tris-egg yolk-glycerol, Tyrode’s albu-
min-lactate-pyruvate (TALP), Cornell University
Extender, sodium citrate-casein, physiological saline
have been used by different workers for evaluating
semen in computer-aided systems (Table 1). Davis and
Boyers (1992) observed increase in velocity measure-
ments like curvilinear velocity (VCL) and straight line
velocity (VSL) with the phosphate buffer saline (PBS) by
10–17% but not with that of homologous seminal
plasma (HSP). However, the percentage of motile sperm
and amplitude of lateral head displacement (ALH) were
found to decrease in both these diluents. Rijsselaere
et al. (2003) evaluated four diluents viz. HEPES TALP,
prostatic fluid, Tris egg yolk glucose and physiological
saline for their effect on different sperm motion charac-
teristics. The velocity parameters (VAP, VSL, VCL) and
the percentage of motile, progressive and rapid sperma-
tozoa were higher for the semen samples diluted in
HEPES-TALP or prostatic fluid in comparison with
other two diluents. Tris-egg yolk and physiological
saline diluted semen samples showed similar measure-
ments for most of the evaluated parameters except BCF,
and percentage of rapid and medium moving sperma-
tozoa were significantly lower (p < 0.05) in tris-egg
yolk extender. Further, velocity measures like VAP and
VCL were the lowest in tris-egg yolk extender among the
four diluents and this could be attributed to higher
viscosity of the diluent leading to slow down of motile
spermatozoa. Therefore, it has to be mentioned that
effect of the diluent on motility characteristics of
spermatozoa needs to be understood clearly before
interpreting the CASA results.
The sperm concentration of semen samples suitable
for evaluation in CASA as reported by various workers
is given in Table 2. Amann (1989) reported that for
computer evaluation of motion characteristics, the
cryopreserved bull semen should be diluted to 10–
12 · 106 ⁄ ml to 40–60 · 106 ⁄ ml. Davis and Katz (1993)
observed the inability of CASA instrument to analyse
the sample when the concentration was greater than
approximately 50 · 106 spermatozoa per ml and less
than approximately 20 · 106 spermatozoa per ml. Most
of the reports indicate that a concentration of approx-
imately 25–30 million sperm cells ⁄ ml as optimal for
motility analysis using CASA (Farrell et al. 1998;
Table 2. Sperm concentration, ex-
tender used and loading volume for Sperm
bull sperm analysis concentration
6
10–12 · 10
40–50 · 106
20–50 · 106
50 · 106
20 · 106
25 · 106
15–30 · 106
25 · 106
30 · 106
30 · 106
30 · 106
TALP, Tyrode’s albumin-lactate-pyruvate; CUE, Cornell University extender.
a
Commercial extender.
‘–’ Not known.
 2010 Blackwell Verlag GmbH
167
Kathiravan et al. 2005; Hoflack et al. 2007). However,
there are reports in which sperm concentration as high
as 40–50 millions per ml was used for analysis (Tuli et al.
1992; Hirai et al. 1997). The loading volume of semen
ranged between 4 (Karthikeya 2003; Kathiravan et al.
2005) and 10 ll (Tuli et al. 1992). The loading volume
was primarily dependent on the sperm concentration,
the extender used and the type of loading chambers
utilized. Normally, a 4–7 ll drop of diluted semen was
used for analysis in Makler’s chamber (Hirai et al. 1997;
Januskauskas et al. 1999; Hoflack et al. 2007), while it
was slightly higher (7–10 ll) when loaded in other
chambers. Tardif et al. (1997) used Microcell chamber
(20 lm deep) with Hamilton Thorne Integrated visual
optical system operated at 37C and loaded 7 ll of
semen diluted approximately to 20 · 106 sperm ⁄ ml.
However, Tuli et al. 1992 loaded 10 ll of semen with a
concentration of 40–50 million spermatozoa per ml
adjusted in a lactose-based extender.
Assessment of Motility Characteristics of Bull
Spermatozoa
The graphical illustration showing the sperm track and
different sperm motion characteristics is given in Fig. 1.
The commonly reported CASA parameters include total
motility, progressive motility, track speed (VCL), path
velocity (VAP), progressive velocity (VSL), amplitude of
lateral head displacement (ALH), beat cross frequency
(BCF), straightness (STR) and linearity (LIN). These
CASA parameters have been modelled and refined
mathematically to describe the motion parameters of
each sperm as it travels through a microscopic field.
Total motility is the ratio of motile cells to the total cell
concentration expressed as percentage. Progressive
motility is the number of cells expressed as percentage
that move with path velocity greater than medium VAP
cut-off and having STR greater than the standardized
threshold. Progressive velocity (VSL) is the straight line
distance between the beginning and the end of the track
divided by the time elapsed. Track speed is a measure of
VCL and it depends on the acquisition rate. Path
velocity (VAP) is defined as the total distance along the
smoothened average path for each cell divided by the
time elapsed. Among these velocity parameters, VCL is
always the highest of the three values while VSL is the
Loading
Extender used volume (ll) Reference
– – Amann (1989)
Lactose-based extenders 10 Tuli et al. (1992)
Phosphate buffer saline 7 Davis and Katz (1992)
Tris-egg yolk-glycerol 5 Hirai et al. (1997)
TALP ⁄ CUE 7 Tardif et al. (1997)
TALP – Farrell et al. (1998)
Glycerolated Triladyl extendera 5 Januskauskas et al. (1999)
Sperm TALP – Amann et al. (2000)
Tris-egg yolk-glycerol 4 Karthikeya (2003)
Tris-egg yolk-glycerol 4 Kathiravan et al. (2005)
Physiological saline 7 Hoflack et al. (2007)
168 P Kathiravan, J Kalatharan, G Karthikeya, K Rengarajan and G Kadirvel
Fig. 1. Sperm motion track showing different motion characteristics
assessed by CASA (VCL, curvilinear velocity; VAP, average path
velocity; VSL, straight line velocity)
lowest. With regular and linear trajectories of sperm
movement, VAP is almost equal to VSL. When the
sperm track is non-linear with high degree of lateral
head movement, then the VAP will be much higher than
the VSL (Mortimer 2000). Straightness measures the
departure of the cell path from a straight line. It is the
ratio of VSL ⁄ VAP. Linearity measures the departure of
the cell track from a straight line. It is the ratio of
VSL ⁄ VCL. The ALH corresponds to the mean width of
the head oscillation as the sperm swims. Beat cross
frequency is determined by measuring the frequency
with which the sperm track crosses the cell path in either
direction. The BCF is a useful value in the estimation of
gross changes in the flagellar beat pattern and depends
on the frame rate of the machine (Mortimer and Swan
1999).
The most important aspect of analysing data from
CASA systems lies very much in understanding the fact
that the estimation of averages of kinematic parameters
cannot be an appropriate approach. The basic reason is
that for most of the kinematic parameters, the population
of sperm cells analysed does not conform to the statistical
normal distribution and hence parametric statistics are
not appropriate methods for data analysis (Holt et al.
2007). The mean values do not offer much insight into the
true structure of the sperm population and hence it is
essential to estimate the proportion of sub-population of
sperm cells with different kinematic patterns.
Sub-population of sperm cells in semen samples
It has been found that different sub-populations of
spermatozoa coexist in most of the mammalian ejacu-
lates (Holt 1996; Abaigar et al. 1999; Quintero-Moreno
et al. 2003, 2007; Cremades et al. 2005; Nunez-Martinez
et al. 2006a; Rivera et al. 2006) including bovines. This
may possibly be attributed to the differences in origin,
assembly of individual spermatozoa and as well as the
differential maturational status and age when they get
mixed in epididymis. The heterogeneity of sperm pop-
ulation in epididymal reserves and subsequently in the
ejaculate can be reflected in differences in morphology,
motility, viability and ultimately in fertilizing capacity of
spermatozoa (Rodriguez-Martinez and Barth 2007).
CASA classifies different sub-population of spermato-
zoa based on path velocity especially low VAP cut-off
(LVV) and medium VAP cut-off (MVV). The overall
sperm population is subdivided into four categories:
Rapid (VAP > MVV); Medium (LVV < VAP <
MVV); Slow (VAP < LVV) and Static (fraction of
cells that are not moving during the analysis). Individual
variation in semen freezability is recognized to exist in
most domestic species, including bovine (Parkinson and
Whitfield 1987) and such variation has been related to
the incidence of distinctly motile sperm sub-populations
present in the fresh ejaculates (Davis et al. 1995; Nunez-
Martinez et al. 2006a,b). The data of different sub-
populations of spermatozoa can be used to predict the
percentage of sperm cells that remain viable after
thawing by establishing a relationship between the
motion and the cells’ resistance to cryopreservation. In
a study on Holstein sperm, significant correlations
(r = 0.473; p < 0.01) were found to exist between the
proportion of highly active and progressive spermatozoa
in the fresh ejaculates and those in the frozen-thawed
semen (Muino et al. 2008) and thus indicating that the
ejaculates with the highest sub-populations of rapid and
progressive sperm were also the most resistant to
cryopreservation. Similarly, the fragmentation of sperm
cell population can be allowed to demonstrate the
differential response of sperm cells to stimulants such as
caffeine and bicarbonate, with some sub-populations
not at all responding to the treatment (Verstegen et al.
2002). Analysis of sperm sub-populations using CASA
can be utilized to differentiate the sperm cell composi-
tion in different age groups of bulls, which may have
some correlation with the differential freezability of
semen from young to old animals (Hallap et al. 2006).
Thus, the detection of these sub-populations may reflect
discrete functional and possibly adaptive differences of
spermatozoa during their genesis and subsequent stor-
age in epididymis. Further, CASA may help in identi-
fying the motion characteristics of potentially fertile
sub-population of spermatozoa in an ejaculate ⁄ insemi-
nate that reach the site of fertilization after surviving the
nature’s rigorous selection process in the female genital
tract (Rodriguez-Martinez 2007).
Hyperactivated motility
Capacitated mammalian spermatozoa exhibit vigorous,
non-progressive, non-linear motion called ‘hyperactiva-
tion’ shortly before or about the time of acrosome
reaction as they progress through the female oviduct.
During hyperactivation, the pattern and vigour of the
sperm track undergo dramatic changes, characterized by
wide amplitude, marked lateral movements of head and
tail, coupled with a slow or non-progressive motility and
‘star-spin’ movement. Hyperactivation is thought to
provide strong thrusting power to the spermatozoa
while passing through the egg investments, particularly
zona (Yanagimachi 1984). In some studies, it has been
shown that sperms that lost their hyperactivated motil-
ity are unable to fertilize the oocytes in vitro (Fleming
and Yanagimachi 1982; Fleming and Kuehl 1985). The
objective measure of this physiological process can serve
as a biological marker to evaluate the functional
capabilities of spermatozoa. However, it had been quite
difficult to assess the hyperactivated spermatozoa in
 2010 Blackwell Verlag GmbH
Motion Analysis of Bull Sperm in CASA – A Review 169

CASA because of poor tracking efficiency. But with the

64.8 ± 1.8
39.5 ± 0.9
89.2 ± 1.9
69.9 ± 1.6
169.2 ± 4.2
9.0 ± 0.2
27.9 ± 0.5
74.8 ± 0.6
48.0 ± 0.7
Rengarajan
Kangayam
B. indicus

(2004)
use of CASA having 60-Hz frame rate and higher
resolution optical system, it has become possible to
evaluate hyperactivated spermatozoa with high veloci-
ties and erratic tracks (Marquez and Suarez 2007). It is
now universally agreed that the distinguishing charac-

B. taurus · Bos indicus

Jersey · Red Sindhi

teristics of hyperactivated spermatozoa are increased

Kathiravan et al.
57.4 ± 4.6
29.1 ± 3.1
93.4 ± 2.9
71.5 ± 2.5
179.8 ± 7.4
9.7 ± 0.5
29.2 ± 0.9
74.4 ± 1.4
40.5 ± 1.6
VCL and ALH along with decrease in LIN. Normally,

(2005)
spermatozoa with VCL higher than 70 lm ⁄ s and ALH
higher than 7 lm are considered as indicative of
hyperactivation (Robertson et al. 1988; Mortimer and
Mortimer 1990; Zhu et al. 1994; Marquez and Suarez
2007).

B. taurus · B. indicus
Post-thaw

Friesian · Sahiwal

Kathiravan et al.
48.2 ± 3.8
24.3 ± 2.3
86.9 ± 2.5
68.1 ± 1.9
172.2 ± 5.4
9.5 ± 0.3
28.1 ± 0.4
76.4 ± 0.8
40.3 ± 0.7
Sperm motion characteristics in different genetic groups

(2005)
Differences in the sperm motility parameters across
different genetic groups of bulls based on subjective
evaluation has been reported earlier (Hoflack et al.
2003). CASA not only allows comparing the sperm
motility across different genetic groups, but also differ-
ent velocity and motion characteristics of spermatozoa.

Hallap et al.
67.2 ± 2.3
36.6 ± 5.1
95.3 ± 1.5
84.5 ± 2.0
163.4 ± 5.4
B. taurus

Holstein
To elucidate the presence of genetic basis for motility

(2006)
differences, it will be highly relevant if similar parameter

–
–
–
–
settings of the equipment are used. In this article, an
attempt has been made to compare the sperm motion
parameters to get a rough estimation of motility 65.4 ± 2.2
35.1 ± 1.7
98.8 ± 2.2
74.8 ± 1.5
184.9 ± 5.5
9.3 ± 0.3
27.6 ± 1.0
73.4 ± 1.0
40.5 ± 0.9
Karthikeya
differences between the genetic groups as the parameter
B. taurus

(2003)
Jersey

set up varied in different studies. Various sperm motion

characteristics of bull spermatozoa in different genetic
groups are shown in Table 3. Hoflack et al. (2007)
compared the motility characteristics of dairy (Holstein-
Holstein- Friesian

Friesian) and beef (Belgian Blue) bull sperms under

Hoflack et al.
82.9 ± 12.0
68.9 ± 11.7
120.9 ± 14.4
109.4 ± 14.2

4.5 ± 0.8
39.0 ± 3.0
89.2 ± 3.1
similar parameter setup of CASA. Substantial differ-
B. taurus

(2007)
Table 3. Mean sperm motion characteristics of bull spermatozoa in different genetic groups

ences in various motility parameters were observed with

–

Holstein-Friesian having better estimates than that of

Belgian Blue cattle. Analysis of sperm sub-populations
revealed fewer rapid and more slow and static sperma-
tozoa being present in Belgian Blue compared to that of
Hoflack et al.
70.4 ± 15.2
53.8 ± 14.8
114.0 ± 18.3
Belgian Blue

Holstein-Friesian. The percentages of total and pro-

98.8 ± 1.8

5.0 ± 1.0
36.9 ± 3.7
84.9 ± 4.0
B. taurus

(2007)

gressively motile spermatozoa and the sperm velocity

–

parameters (VAP, VSL and STR) were significantly

Pre-freeze

lower in the Belgian Blue breed. Further, lower kinetic

efficiency of Belgian Blue cattle was observed in terms of
lower BCF and higher ALH. A similar pattern of
95.9 ± 0.5
54.1 ± 1.9
127.1 ± 3.4
88.4 ± 1.9
239.3 ± 6.1

25.7 ± 0.8
69.2 ± 1.2
38.1 ± 0.8
Karthikeya
10.4 ± .2
B. taurus

motion characteristics (lower progressive motility, VSL,

(2003)
Jersey

BCF and higher ALH) was observed in case of Jersey

(Bos taurus) and Kangayam (Bos indicus) bulls (Karthi-
keya 2003; Rengarajan 2004), although the parameter
set up varied in the latter studies. Regarding the post-
Farrell et al.

thaw sperm motion characteristics, progressive motility

Bos taurus

(1998)
5
89
70

15
92
88
138
127
144

and velocity were higher in Holstein bulls (Hallap et al.

–

2006) than that of Jersey breed. However, most of the

parameters were found to be lower in crossbred cattle
(B. taurus · B. indicus) and Kangayam (B. indicus)
cattle except ALH, BCF and STR (Kathiravan et al.
Progressive velocity (lm ⁄ s)

Beat cross frequency (Hz)

Progressive motility (%)

2005).
Lateral amplitude (lm)
Path velocity (lm ⁄ s)

Track speed (lm ⁄ s)

Total motility (%)

Straightness (%)

Significance of Different Sperm Motion

Genetic group

Linearity (%)

Characteristics in Predicting Bull Fertility

Reference

Motility is commonly believed to be one of the most

Breed

important characteristics associated with the fertilizing

 2010 Blackwell Verlag GmbH

170 P Kathiravan, J Kalatharan, G Karthikeya, K Rengarajan and G Kadirvel
ability of sperm. Significant differences had been
observed in motility parameters between sperm that
achieved a high percentage of fertilization and those that
failed in the achievement of pregnancy (Donnelly et al.
1998). The relationship between subjectively evaluated
sperm motility and fertility are contradictory in nature
with some reports (Wood et al. 1986; Kjaestad et al.
1993; Correa et al. 1997; Zhang et al. 1998) demon-
strating the significant relationship between them, while
others were not able to establish such a relationship
(Soderquist et al. 1991; Andersson et al. 1992; Janusk-
auskas et al. 1999). The lack of significant relationship
between motility and fertility might be attributed to
several causes that include variation between individual
bulls, presence of excess ⁄ adequate number of sperma-
tozoa in inseminates (Gerard and Humblot 1991;
Januskauskas et al. 1996), etc. However, CASA gives a
wide range of sperm motility parameters that provide
more valuable information on the physiological status of
spermatozoa and thus has the potential for a more
accurate prediction of fertility than the parameters
assessed by the routine microscopical semen evaluation
(Farrell et al. 1998; Christensen et al. 1999). Among the
different motion characteristics assessed by CASA, some
may be functionally significant from fertilization point
of view. Many researchers have tried to correlate
different motion characteristics of bull spermatozoa
with oocyte penetration rate, in vitro fertility rate and
field fertility (Farrell et al. 1998; Kathiravan et al. 2008).
Budworth et al. (1987, 1988) observed significant
correlations of the percentage of motile sperm and
spermatozoon velocity with the competitive fertility
index. Amann (1989) studied the motility characteristics
of the frozen, thawed bull spermatozoa and observed a
correlation of 0.86, 0.68, 0.70, 0.60, )0.05 and )0.16 for
per cent motile spermatozoa, VCL, VSL, LIN, ALH and
BCF respectively with competitive fertility index. Farrell
et al. (1998) reported very high correlation between
combined motility parameters measured by CASA on
fresh semen and bull fertility with good predictive value
(r2 = 0.63–0.98), when fertility was defined as 59 days of
non-return to service. Similarly, Januskauskas et al.
(2000) reported significant correlation between per cent
linearly motile spermatozoa with 56-day non-return rate
in Swedish Red and White dairy bulls. Kathiravan et al.
(2008) assessed the relative efficacy of different motion
characteristics of bull spermatozoa to predict oocyte
penetration rate in zona-free hamster oocytes and found
highly significant (p < 0.01) positive correlation
(r = 0.791) between fertilization percentage and pro-
gressive motility. Among different CASA variables,
progressive motility alone contributed to 62.6% and
together with membrane integrity, it contributed to
64.1% of variation in the fertilization percentage. The
velocity measurements (VAP and VSL) had significant
effect on in vitro fertility and contributed only 41.2% of its
variance, while they contributed to 66.1% of variation
along with progressive motility and per cent hypo-
osmotic reacted spermatozoa.
All these studies showed that among the different
sperm motion characteristics assessed by CASA, pro-
gressive motility and velocity parameters like VCL, VSL
and path velocity (VAP) can be of use in predicting the
fertility of bull semen. Significant positive correlations
between different velocity parameters and fertilization
percentage obtained in bull sperm are similar to the
reports made in human spermatozoa (Fetterholf and
Rogers 1990; Liu et al. 1991). However, parameters like
STR, LIN, BCF and ALH were found to have almost
no correlation with bull fertility in these studies, which is
in contrast to the positive correlation between some of
these parameters and in vitro fertilization rates of human
spermatozoa (Sukcharoen et al. 1995, 1998).
Conclusion
CASA has been demonstrated to be a useful tool to
objectively analyse the sperm head movement and effect
of different treatments on sperm kinematics. The ability
of CASA to classify different sperm sub-populations
provides an opportunity to establish criteria for different
functional aspects of spermatozoa related to fertility,
such as ability to penetrate cervical mucus, to undergo
capacitation and acrosome reaction, penetration of
cumulus, zona-binding. However, there has been a lack
of uniformity among users and in defining universally
accepted values for normal and abnormal sperm
motion. Hence, to refine the technique, standardization
and quality control are the absolute pre-requisites. Basic
definition studies need to be performed to set up the
limits, taking into account in particular species and age
variations. Also, further investigation is needed to know
the scientific basis for the ability of different sperm
motion characteristics in predicting the fertility rate. In
spite of the above needed improvements, CASA can be
considered as an efficient, precise and reliable tool to
evaluate fertility as well as to evaluate the physiological
differences in sperm motion characteristics in different
genetic and age groups.
Conflict of Interest
None of the authors have any conflict of interest to declare.
Author contributions
All the authors have the experience of working on sperm cryo-
preservation and evaluation, especially in sperm evaluation using
computer-aided system. Kathiravan and Kalatharan were involved in
conceptualizing the article. Kathiravan, Karthikeya and Rengarajan
were involved in collecting literatures and drafting the manuscript.
Kalatharan and Kadirvel critically reviewed the manuscript. Kadirvel
was involved in the revision process of the manuscript.
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Submitted: 18 Sep 2008; Accepted 10 Feb 2009
Author’s address (for correspondence): Dr P Kathiravan, National
Bureau of Animal Genetic Resources, P.B. No 129, GT Road Bypass,
Karnal 132001, Haryana, India. E-mail: kathirvet@yahoo.co.in
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